Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein

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Abstract

A specific binding site for the antiepileptic drug levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra®) in rat brain, referred to as the levetiracetam binding site, was discovered several years ago. More recently, this binding site has been identified as the synaptic vesicle protein 2A (SV2A), a protein present in synaptic vesicles [Lynch, B., Lambeng, N., Nocka, K., Kensel-Hammes, P., Bajjalieh, S.M., Matagne, A., Fuks, B., 2004. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc. Natl. Acad. Sci. USA, 101, 9861–9866.]. In this study, we characterized the binding properties of levetiracetam in post-mortem human brain and compared them to human SV2A expressed in Chinese hamster ovary (CHO) cells. The results showed that the binding properties of levetiracetam and [3H]ucb 30889, an analogue that was previously characterized as a suitable ligand for levetiracetam binding site/SV2A in rat brain [Gillard, M., Fuks, B., Michel, P., Vertongen, P., Massingham, R. Chatelain, P., 2003. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur. J. Pharmacol. 478, 1–9.], are almost identical in human brain samples (cerebral cortex, hippocampus and cerebellum) and in CHO cell membranes expressing the human SV2A protein. Moreover, the results are also similar to those previously obtained in rat brain. [3H]ucb 30889 binding in human brain and to SV2A was saturable and reversible. At 4 °C, its binding kinetics were best fitted assuming a two-phase model in all tissues. The half-times of association for the fast component ranged between 1 to 2 min and represent 30% to 36% of the sites whereas the half-times for the slow component ranged from 20 to 29 min. In dissociation experiments, the half-times were from 2 to 4 min for the fast component (33% to 49% of the sites) and 20 to 41 min for the slow component. Saturation binding curves led to Kd values for [3H]ucb 30889 of 53 ± 7, 55 ± 9, 70 ± 11 and 75 ± 33 nM in human cerebral cortex, hippocampus, cerebellum and CHO cells expressing SV2A respectively. Bmax values around 3–4 pmol/mg protein were calculated in all brain regions. Some of the saturation curves displayed curvilinear Scatchard plots indicating the presence of high and low affinity binding sites. When this was the case, Kd values from 25 to 30 nM for the high affinity sites (24% to 34% of total sites) and from 200 to 275 nM for the low affinity sites were calculated. This was observed in all brain regions and in CHO cell membranes expressing the SV2A protein. It cannot be explained by putative binding of [3H]ucb 30889 to SV2B or C isoforms but may reflect different patterns of SV2A glycosylation or the formation of SV2A oligomers. Competition experiments were performed to determine the affinities for SV2A of a variety of compounds including levetiracetam, some of its analogues and other molecules known to interact with levetiracetam binding sites in rat brain such as bemegride, pentylenetetrazol and chlordiazepoxide. We found an excellent correlation between the affinities of these compounds measured in human brain, rat brain and CHO cells expressing human SV2A. In conclusion, we report for the first time that the binding characteristics of native levetiracetam binding sites/SV2A in human brain and rat brain share very similar properties with human recombinant SV2A expressed in CHO cells.

Introduction

Levetiracetam (2S-(oxo-1-pyrrolidinyl)butanamide, Keppra®) is a novel antiepileptic drug marketed as add-on therapy for partial-onset seizures in adults (Hovinga, 2001).

Levetiracetam has a peculiar pharmacological profile that distinguishes it from all the other antiepileptic drugs. While it affords protection against seizures induced in audiogenic genetically susceptible mice as well as in electrically orpentylenetetrazol induced kindling models, it is devoid of anticonvulsant activity in most acute animal models of epilepsy such as the maximal electroshock model (Klitgaard et al., 1998, Klitgaard, 2001). Its mechanism of action does not involve the classical targets of the other antiepileptic drugs i.e. γ-aminobutyric acid (GABA) facilitation, inhibition of sodium channels or modulation of low-voltage activated calcium channels (Margineanu and Klitgaard, 2002, Rigo et al., 2002). However, a brain specific binding site for levetiracetam, named levetiracetam binding site was described and characterized in rat brain for the first time in 1995 (Noyer et al., 1995) using [3H]levetiracetam as radioligand. The binding of levetiracetam to this site was reversible, saturable and highly stereoselective. The availability of a new, more potent analogue, [3H]ucb 30889 enabled further characterization of these sites using autoradiography (Gillard et al., 2003). Also, irreversible binding of the radioligand under UV irradiation was performed permitting covalent binding and subsequent determination of the molecular weight of the binding site (Fuks et al., 2003). This led to the cloning and the identification of this site as being the SV2A protein (Lynch et al., 2004). The combined observations that SV2A knock-out mice develop lethal seizures soon after birth (Crowder et al., 1999) together with an excellent correlation between the affinity of levetiracetam analogues for SV2A in rat brain or for the recombinant protein and their anticonvulsant potency in audiogenic mice (Noyer et al., 1995, Lynch et al., 2004) strongly support a role of SV2A in the antiepileptic properties of levetiracetam.

The biology of SV2A itself is not entirely elucidated but it has been proposed that SV2 proteins are involved in exocytosis and more particularly, because of their structural analogy with transporters, may play a role in the homeostasis of synaptic vesicle constituents such as ATP or calcium (Bajjalieh et al., 1992, Janz et al., 1999). Impact on calcium homeostasis is further supported by data showing that SV2A interacts with synaptotagmin, a protein considered as a calcium sensor (Schivell et al., 2005) and by the recent publication by Cataldi et al. (2005) showing that levetiracetam inhibits bradykinin- and ATP-induced inositol triphosphate (IP3)-dependent calcium release in PC12 cells, cells that are known to possess SV2A but not in 3T3-fibroblasts where SV2A is absent.

This paper reports an in-depth comparison of the binding properties of the human recombinant SV2A protein expressed in CHO cells with those of the native protein, present in its natural environment, the human brain.

Section snippets

Drugs and radioligand

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide), piracetam (2-(2-oxo-1-pyrrolidinyl)acetamide), ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide) and analogues were synthesised at UCB (Braine L'Alleud, Belgium). Bemegride, bicuculline, chlordiazepoxide and pentobarbital were purchased from Sigma-Aldrich (Bornem, Belgium). Pentylentetrazol was acquired from Acros Organics (Pittsburgh, USA). [3H]ucb 30889 (32 Ci/mmol) was custom labelled by Amersham Biosciences

Binding kinetics

The binding of [3H]ucb 30889 in human brain and to recombinant SV2A was reversible and displayed both biphasic association and dissociation kinetics as depicted in Fig. 1 and reported in Table 1. The fast component for both association and dissociation had a half-time ranging from 1 to 4 min and represented from 30% to 36% of the sites in association experiments and from 33% to 49% in dissociation experiments. The half-times of the slow component ranged from 20 to 41 min.

The dissociation

Discussion

The antiepileptic drug levetiracetam has been shown to bind to a specific binding site in rat brain (Noyer et al., 1995, Gillard et al., 2003). This binding site has been identified as the SV2A protein, a protein ubiquitously distributed in the central nervous system as a component of synaptic vesicles (Lynch et al., 2004). In our study, we aimed to extend these observations by studying more in depth the binding properties of [3H]ucb 30889 in CHO cells stably expressing the human recombinant

Acknowledgements

The authors wish to thank Mrs. Isabelle Jacques, Mr. Frédéric Aelbrecht and Mr. Michel Legrand for their skilful technical assistance.

We also want to acknowledge Dr Berkley Lynch and Mrs. Dina Anderson for providing us with the CHO clone expressing the human SV2A protein.

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